62, 1^ Correspondence^ 14310. .11, B., PERANI, E. G. and GROSSET, J.-H. Effectivenessof clarithromycin and minocycline aloneand in combination against experimental Mycobacteriumleprac infection in mice. Antimicrob.Agents Chemother. 35 (1991) 579-581.11. Ji, B., PERANI, E. G., PETINON, C. and GROSSET,J. H. Bactericidal activities of single or multipledoses of various combinations of new antileprosydrugs and/or rifampin against M. leprae in mice.Int. J. Lepr. 60 (1992) 556-561.12. SHEPARD, C. C. Statistical analysis of results obtainedby two methods for testing drug activityagainst Mycobacterium Ieprae. Int. J. Lepr. 50(1982) 96-101.13. SHEPARD, C. C., REES, R. J. W., LEVY, L., PATTYN,S. F., J1, B. and DELA CRUZ, E. C. Susceptibilityof strains of Mycobacterium Ieprae isolated priorto 1977 from patients with previously untreatedlepromatous leprosy. Int. J. Lepr. 54 (1986) I I —15.14. WALKER, L. L., VAN LANDINGHAM, R. M. andSHINNICK, T. M. Clarithromycin is bactericidalagainst strains of Mycobacterium Ieprae resistantand susceptible to dapsone and rifampin. Int. J.Lepr. 61 (1993) 59-65.Active Humoral Immunity in the Absence of Cell-MediatedImmunity in Murine Leprosy: Lastly an ExplanationTo THE EDITOR:Until recently, a puzzling phenomenon in(human) lepromatous leprosy was that relatedto the patients' immune responsivenessto antigens of Mycobacterium Ieprae.It is a well-accepted fact that lepromatouspatients show an absence of cell-mediatedimmunity (CMI) to M. leprae antigens whilethey retain unaltered their humoral (antibody-mediated)immunity (AbMI). This waspuzzling, because the great majority of themicrobial antigens belong to the so-called"thymus dependent" type, i.e., they needthe participation of T-helper lymphocytes(LcTh) to generate efficient antibody responses.How, then, would the dramatic alterationin the T-cell-mediated immunecompetence of the host not reflect on itshumoral competence?An analogous situation of a gradual lossof CMI with an apparently unaltered AbMIhas been found in mice suffering from "murineleprosy," a disease caused by Al. lepraemurium(MLM) and characterized by thedevelopment of granulomatous lesions inthe skin and viscera that highly resemblethe lepromatous lesions of human leprosy.In both mycobacterioses, several investigationspoint to a deficit in the functionof the helper population of T lymphocytesdue to the absence of antigen-reactive T cells,the lack of interleukin-2 (IL-2)-producingcells, the excess of suppression by suppressorT (CD8+ or Ly2 9 3 +) cells, the suppressionby macrophage-derived factors, etc. (fora recent review on the subject, see 1 '). Thosesteps within the intricate net of cellular interactionsthat have been found altered inhuman lepromatous or mouse lepromatoidleprosy and that could, to a certain extent,explain the finding in both diseases of depressed(or absent) CMI to the mycobacterialantigens, are illustrated in an oversimplifiedmanner in Figure 1.For a long time, in the mouse as well asin humans the existence and function of onlyone class of helper (L3T4+/CD4+) T lymphocyte(a Th lymphocyte able to mediatecellular immunity and, at the same time,able to cooperate with B lymphocytes forantibody production) was accepted (Fig. 2).The existence of a "bifunctional" T cell,however, does not help to explain the persistenceof an active humoral immunity inthe absence of a helper CMI to the mycobacterial(thymus-dependent) antigens.On the other hand, the still recent discoveryof two subpopulations of helper(L3T4+) T cells in the mouse (Th 1 and Th2)(2 • 3. 9, 10‘ allows one to tentatively explainthe apparent paradox mentioned above.Both Th 1 and Th2 clones produce and releaseIL-3, GM-CSF, TNF-a, in addition toother proteins. However, while TH 1 clonessecrete IL-2, interferon--y and lymphotoxin,Th2 clones secrete IL-4, IL-5, IL-6, and IL-
144^ International Journal of Leprosy^ 1994LYMPH NODESMLr-TcellTaePROLIFERATIONLc CD 4+0LYMPHOKI NESIL- 2SUPPRESSION: .SUPPRESSION 4 4 :TS^Io^v. Ag-SFII:111 *•MAF, IFN• MPH ACTIVATIONAg+SF^)) TST Lc VV` CD8 +(CS cells)FIG. 1. Cell interactions in cell-mediated immunity, showing those steps that have been found altered (ornormal in contradictory reports) in lepromatous leprosy. 0 availability of circulating Al. frprue-reactive (MLr)T cells; some authors suggest that a great amount of lymphocytes arc trapped into the lymph nodes, leading tothe lowering of their levels in circulation; 0 antigen handling and/or interleukin-1 (IL-1) production by macrophages(MPH), 0 synthesis and release of IL-2 by MLr T cells; e expression of IL-2 receptors (Tac) by Thelper/inducer cells; 0 IL-2/antigen-driven proliferation of T cells with concomitant synthesis and release oflymphokines (depending on steps I to 4); a bactericidal response of macrophages to the infecting mycobacteria;0 lymphokine-dependent activation of macrophage; ® suppressive effects of mycobacterial components andfactors released from bacilli-laden macrophages, ® ML-induccd activation of CD8+ T (suppressor) cells (TS)able to produce antigen-specific suppressor factors (Ag + SF), and Ag + SF-dependent activation of third-partylymphocytes able to produce nonspecific suppressor factors (Ag-SF); 0 downregulation by Vida rrttosa ("contrasuppressor")cells (CS) (see " for a more detailed description of this figure).10. It has been proposed that although Th 1cells may cooperate with B cells, their mainroll is related to lymphokine production,macrophage activation and delayed hypersensitivity( 2). The Th2 clones, on the otherhand, are more cooperative with B cells ( 14 ).Thl cells and Th2 cells seem to be mutuallyinhibitory (Th I -derived IFN-y inhibits thein vitro proliferation of Th2, and Th2-derived IL-4 or IL-10 might inhibit the proliferationof Thl clones ( 7 ). Since inadvanced murine leprosy CMI to MLM antigensis absent but humoral immunity isnot, it is possible that Th2 cells may be thepredominant (at least functionally) subpopulation.Thus, contrary to Figure 2, Figure3 takes into consideration the participationof two subclasses of T helper lymphocytesand explains, reasonably well, the depressedor absent cellular immunity in the presenceof a hyperactive antibody-mediated immunity.This is the situation reported repeatedlyin leprosy. On activation (in an Ia-restrictedmanner), Th 1 lymphocytes produce IL-2and respond to it, to eventually proliferateand give rise to the elements of CMI anddelayed hypersensitivity. It is through solublefactors (such as IFN-y) that Thl lymphocytesactivate macrophages increasing
62, 1^ Correspondence^ 145Th2 RESPONSEOr-0[CELL MEDIATED IMMUNITY ILyinpokille, productionMacrophage activationrav-It2G2 (1111)-m-101,1gE (Thu)Thl RESPONSELIUMDRAL IMMUNITY f Abl IIHUMORAL IMMUNITY^ISC. "Suppressor cell"H-= SuppressionFIG. 2. A T lymphocyte acts as a bifunctional cell,both directly responsible for CMI and also able to cooperatewith B cells for antibody production (all isotypesincluded). Here, the main regulatory mechanismis exerted by a suppressor cell, either a lymphocyte, amacrophage, or some other cell. The absence of specificT cells would lead to the lack of a CMI response to agiven antigen and to a parallel fall in antibody responseto that antigen.their microbicidal capabilities (l 4). An excessof 1FN--y may, however, suppress thefunction of Th2 lymphocytes ( 6). ActivatedThl lymphocytes seem to cooperate with Bcells to induce the synthesis of IgM, IgG3and IgG2. Activated Th2 cells, to the contrary,produce among other factors IL-4,IL-5 and IL-10. IL-4 will suppress the activationof Thl cells at the same time thatit promotes differentiation and activation ofB cells. B cells activated through Th2 cellsproduce IgM, IgG3, IgG1 and IgE ( 13). Bcells and macrophages function as APC forboth Thl and Th2 cells. Although able tocooperate with B cells, Th 1 cells are lesscooperative than Th2 cells ( 8 ).Since in lepromatous (human and murine)leprosy, there is a gradual loss of CMIthat is inversely proportional to the degreeof antibody response to the mycobacterialantigens, it would seem that initially Th 1cells are the predominantly active T lymphocytes,while the predominant cell populationin the advanced disease are the Th2lymphocytes.Although the mycobacterial epitopes thatstimulate each T-cell subpopulation remainto be identified, it would seem that the immunodominantepitopes (both in humanlepromatous and murine leprosy) are thosethat trigger the Th2 (or human equivalent)(') cells, rather than those that trigger the---e- Suppression / InhibitionFIG. 3. Involvement of two T cells (with the helper/inducer phenotype), one engaged in CMI, the other ableto potentiate the activity of B cells, and both mutuallyinhibitory, explains reasonably well the situation observedin leprosy of deficient or absent CMI to themycobacterial antigens in the presence of normal abilityto produce antibodies to the same antigens.Thl (or human counterpart) (') cells. Accordingly,other epitopes would preferentiallyactivate the Thl cells in tuberculoidleprosy (if there is any) in the mouse. Wehave demonstrated that early in the infectionwith Al. lepraennirizn, the granulomatouslesions are made up of biochemicallyactivated macrophages intermixed withlymphocytes (a tuberculoid-type granuloma).Then, in the late stages of the disease,the macrophages' biochemical activationsubsides and the lesion acquires the characteristicsof lepromatous granulomas (inertand highly bacilliferous macrophages) ( 12 ).Thus, it is possible that activation of Thland Th2 lymphocytes (or the human equivalents)may occur in sequence but, so far,neither the events that drive such a sequencenor the underlying regulatory mechanismsare well understood.Additionally, other factors more thanmere mycobacterial epitopes might alsoparticipate in the differential triggering ofeach subpopulation of L3T4+ cells: inadequateparticipation of APC, whether amacrophage or a B lymphocyte (see below);diverse factors (cytokines) might also tilt thebalance in favor of either the Thl or theTh2 subpopulation (5); some genetic factors(not necessarily MHC-related) might alsoparticipate in the turning on/off of the Thlor Th2 cells, etc. At the moment, all of thesefactors are ill-defined and, hence, they aredifficult to evaluate in vivo. The use ofmonoclonal antibodies to several known interleukinswould permit the identification
146^ International Journal of Leprosy^ 1994ABantigenic fragmentsIL-1IL-1(CB1(as APC)I` C MI^-0-- 1, A bMI(as effector cell)inhibition / suppression. lack ofFIG. 4. A = Functional B cells (B) and macrophages(Mph), acting as antigen-presenting cells (APC) stimulateboth the CMI-commited lymphocytes (Thl) andthose that cooperate with B cells for increased productionof antibodies (Th2). It is proposed that at anearly stage of the infection, Th I cells receive moreinductive stimulation that do Th2 cells, favoring theCMI response and the suppression of Th2 cells, withthe consequent lowering of the antibody-mediated response.Theoretically, B cells are not efficient APC toTh2 lymphocytes because Th2 cells require, in addition,the stimulatory effect of IL-1 and B cells do notproduce significant amounts of it. B = As a consequenceof the infection, B cells might deteriorate, losingtheir ability to function as APC. this would diminishthe activity of Thl cells (decreasing CMI) without alteringthe capacity of Th2 cells to respond to the stimulatoryeffects of macrophages. Activated Th2 cellswould further inhibit the functioning of Th I cells (andCMI) while retaining their potentiating effect on B lymphocytes.and measurement of the IL-2 and IL-6 (orIL-4) levels in the serum of infected individualsand the localization of IL-2-producing(Th 1) and IL-6-producing (Th2)lymphocytes in the lesions, so their participationin the immunopathology of the diseasemight possibly be assessed. Alternatively,Thl and Th2 cells in the lesions alsocould be identified by Northern hybridizationof poly(A) + mRNA isolated from thespleen and lymph nodes at variable timesafter infection with M. lepraenturiton withprobes coding for IL-4, IL-6, IL-10, IL-2,and IFN-y, in the way that has been donefor mice infected with Leishniania major( 7), or by the more practical and sensitivepolymerase chain reaction (PCR) withprobes for the above lymphokines. Also, althoughboth Th 1 and Th2 cells might beable to induce secretion of IgM and IgG3immunoglobulins, IgG 1 (and IgE) secretionseems to be induced solely by Th2 cells andIgG2a solely by Th I cells ( 4). Serologicalidentification and measurement of theseimmunoglobulin isotypes would allow oneto decide which L3T4+ is the dominantparticipant at a given stage of the disease.The regulation of the immune responseby the participation of B lymphocytes asAPC is a possibility that deserves furtherconsideration since these cells can efficientlypresent antigen to T cells without producingsignificant amounts of IL-1. This means thatB cells functioning as APC would activatemainly or solely those Th cells that are IL-1-independent (Th 1 cells). On activation,these cells would produce INF-y which, inturn, might suppress the function of the Th2subpopulation (6), leading to the overalllowering of the antibody response. On theother hand, a defective or a short-lived APCfunction of B cells would not efficiently promoteactivation of lymphocytes, whether ornot IL-1-dependent. The APC function insteadwill be exerted by the constitutivelyIL-1-producing macrophages. Activation ofT cells through macrophages will then favoractivation of the Th2 subpopulation, withthe consequent increase in antibody responseand the simultaneous inhibition ofthe Thl cell subpopulation. Thus, it is proposedthat in those animals that developtuberculoid-like disease, the antigen-presentingmacrophages initiate the response,allowing the activation of both IL-1-dependent(Th2) and IL-1-independent (Thl)lymphocytes. As B cells start functioning asAPC, they will favor activation of IFN-yproducing(IL-1-independent) Th 1 cells.IFN-y would inhibit the function of Th2cells, leading to the lowering of the antibodyresponse (Fig. 4A).In those animals with lepromatoid leprosy,B cells might not persist or even appearas APC. With this function carried out solelyby macrophages, activation of IL-1-dependentTh2 cells would be the one to besupported, and their resulting products (IL-4
62, 1^ Correspondence^ 147and IL-10) would eventually shut off activationof Th 1 cells; this would result in thedepression of CMI and the sustained productionof antibodies (Fig. 4B). At the moment,because the above proposition ismerely speculative, the reasons why B cellsmight not appear or persist as APC are alsospeculative. One way to test the hypothesis,however, could be the administration offunctional, virgin or educated, B cells fromnormal or tuberculoid animals into animalsin which the presence of inefficient (or theabsence of) antigen-producing B cells is suspected;this could retard or even revert thelepromatoid evolution of the disease. Onthe other hand, administration of lepromatoid-derivedB cells to tuberculoid animalscould perhaps drive the course of theinfection toward the lepromatoid pole.—Oscar Rojas-Espinosa, Sc.D.Departanzento de IninunologiaEscuela Nacional de Ciencias 13iologicasInstituto Politécnico NacionalCarpio y Plan de AyalaColonia Santo Tomas11340 México, D.E., MéxicoAcknowledgment. The author thanks Dr. L. JimenezZamudio for his enlightening discussions on thecontents of this essay. The author holds a fellowshipfrom the Instituto Politécnico Nacional, Mexico, fromthe Comisión de Operación y Fomento de las ActividadesAcadémicas del I.P.N, and from the SistemaNacional de Investigadores, Mexico.REFERENCES1. BLOOM, B. R., MODLIN, R. L. and SALGAME, P.Stigma variations—observations on suppressorT-cells and leprosy. Ann. Rev. Immunol. 10 (1992)453-488.2. CHER, D. J. and MOSMANN, T. R. Two types ofmurine helper T cell clone. II. Delayed-type hypersensitivityis mediated by Th I clones. J. Immunol.138 (1987) 3688-3694.3. CHERWINSKI, H. M., SCHUMACHER, J. H., BROWN,K. D. and MOSMANN, T. R. Two types of mousehelper T cells clone. III. Further differences in lymphokinesynthesis between Thl and Th2 clonesrevealed by RNA hybridization, functionallymonospecific bioassays, and monoclonal antibodies.J. Exp. Med. 166 (1987) 1229-1244.4. COFFMAN, R. L., SEYMOUR, B. W., LEBMAN, D. A.,HIRAKI, D. D., CHRISTIANSEN, J. A., S/IRADER, B.,CHERWINSKI, H. M., SAVELKOUL, H. F., FINKEL-MAN, F. D., BOND, M. W. and MOSMANN, T. R.The role of helper T cell products in mouse B celldifferentiation and isotype regulation. Immunol.Rev. 102 (1988) 5-28.5. FERNANDEZ-BOTRAN, R., SANDERS, V. M.,MOSMANN, T. R. and VITETTA, E. S. Lymphokine-mediatedregulation of the proliferative responseof clones of T helper 1 and T helper 2 cells.J. Exp. Med. 168 (1988) 543-558.6. GAJEWSKI, T. F. and FITCH, F. W. Anti-proliferativeeffect of IFN-gamma in immune regulation.I. IFN-gamma inhibits the proliferation of Th2but not Th 1 murine helper T lymphocyte clones.J. Immunol. 140 (1988) 4245-4252.7. HEINZEL, F. P., SADICK, M. D., HOLADAY, B. J.,COFFMAN, R. L. and LOCKSLEY, R. M. Reciprocalexpression of interferon gamma or interleukin 4during the resolution or progression of murincleishmaniasis; evidence for expansion of distincthelper T cell subsets. J. Exp. Med. 169 (1989) 59-72.8. KILLAR, L., MACDONALD, G., WEST, J., WOODS,A. and BorromTY, K. Cloned, la-restricted T cellsthat do not produce interleukin 4(IL-4)/B cellstimulatory factor I (BSF-1) fail to help antigenspecificB cells. J. Immunol. 138 (1987) 1674-1679.9. MOSMANN, T. R., CHERWINSKI, H., BOND, NI. W.,GIEDLIN, M. A. and COFFMAN, R. L. Two typesof murine helper T cell clone. I. Definition accordingto profiles of lymphokine activities andsecreted proteins. J. Immunol. 136 (1986) 2348-2357.10. MOSMANN, T. R. and COFFMAN, R. L. Heterogeneityof cytokine secretion patterns and functionsof helper T cells. Adv. Immunol. 46 (1989)111-147.11. ROJAS-ESPINOSA, 0. and ESTRADA-PARRA, S. Immunologyof leprosy: cellular anergy to Mycobacteriumleprae. Arch. Invest. Med. 20 (1989) 335-341.12. ROJAS-ESPINOSA, 0., VEGA, R., OLTRA, A., ARCE,A. and Nu&Ez, A. Transitory macrophage activationin the granulomatous lesions of Mycobacteriumlepraemurium-induced lepromatoid leprosyin the mouse. Int. J. Lepr. 56 (1988) 428-436.13. STEVENS, T. L., BOBBIE, A., SANDERS, V. M.,FERNANDEZ-BOTRAN, R., COFFMAN, R. L.,MOSMANN, T. R. and VITETTA, E. S. Regulationof antibody isotype secretion by subsets of antigenspecifichelper T cells. Nature (Lond.) 334 (1988)255-258.14. STOUT, R. D. and BOYMMLY, K. Antigen specificactivation of effector macrophages by IFN--y-producing(ThI) T cell clones; failure of IL-4-producing(Th2) T cell clones to activate effector functionin macrophages. J. Immunol. 142 (1989) 760-765.